LOCUS       sequence-434403-        6602 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Reporter plasmid that expresses GFP and firefly luciferase linked 
            with a viral 2A peptide. A stop codon is inserted at the 3'-end of 
            the gfp gene, which permits GFP, but not luciferase expression..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6602)
  AUTHORS   Nemudraia A, Nemudryi A, Wiedenheft B
  TITLE     Repair of CRISPR-guided RNA breaks enables site-specific RNA 
            excision in human cells.
  JOURNAL   Science. 2024 Apr 25:eadk5518. doi: 10.1126/science.adk5518.
  PUBMED    38662916
REFERENCE   2  (bases 1 to 6602)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Science. 
            2024 Apr 25:eadk5518. doi: 10.1126/science.adk5518."
COMMENT     Sequence Label: pAN013
FEATURES             Location/Qualifiers
     source          1..6602
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(268..294)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(310..333)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      333..342
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(425..628)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(454..474)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        629..932
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     rep_origin      complement(1107..1695)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1187..1206)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    2024..2071
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     2096..2115
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(2303..3097)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             complement(2303..3097)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2414..2433)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     3024..3043
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(3132..3489)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3146..3281
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        3298..3489
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     3446..3466
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        complement(3491..3595)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3622..4077
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3709..3728)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3919..3940
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    4084..4205
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4095..4114)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4149..4168
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(4329..5981)
                     /codon_start=1
                     /gene="luc"
                     /product="firefly luciferase"
                     /label=luciferase
                     /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
                     HIEVNITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
                     ANDIYNERELLNSMNISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
                     MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
                     RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
                     IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
                     GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
                     GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
                     LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
                     FVDEVPKGLTGKLDARKIREILIKAKKGGKSKL"
     primer_bind     5897..5915
                     /label=LucNrev
                     /note="Firefly luciferase, reverse primer"
     CDS             complement(5988..6041)
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             complement(join(6186..6602,1..249))
                     /codon_start=1
                     /product="green fluorescent protein 2 from Pontellina 
                     plumata, also known as ppluGFP2 (Shagin et al., 2004)"
                     /label=CopGFP
                     /translation="MPAMKIECRITGTLNGVEFELVGGGEGTPEQGRMTNKMKSTKGAL
                     TFSPYLLSHVMGYGFYHFGTYPSGYENPFLHAINNGGYTNTRIEKYEDGGVLHVSFSYR
                     YEAGRVIGDFKVVGTGFPEDSVIFTDKIIRSNATVEHLHPMGDNVLVGSFARTFSLRDG
                     GYYSFVVDSHMHFKSAIHPSILQNGGPMFAFRRVEELHSNTELGIVEYQHAFKTPIAFA
                     "
     CDS             complement(join(6186..6602,1..249))
                     /codon_start=1
                     /product="green fluorescent protein 2 from Pontellina 
                     plumata, also known as ppluGFP2 (Shagin et al., 2004)"
                     /label=CopGFP
                     /translation="MPAMKIECRITGTLNGVEFELVGGGEGTPEQGRMTNKMKSTKGAL
                     TFSPYLLSHVMGYGFYHFGTYPSGYENPFLHAINNGGYTNTRIEKYEDGGVLHVSFSYR
                     YEAGRVIGDFKVVGTGFPEDSVIFTDKIIRSNATVEHLHPMGDNVLVGSFARTFSLRDG
                     GYYSFVVDSHMHFKSAIHPSILQNGGPMFAFRRVEELHSNTELGIVEYQHAFKTPIAFA
                     "
ORIGIN
        1 gtagccgccg ttgttgatgg cgtgcaggaa ggggttctcg tagccgctgg ggtaggtgcc
       61 gaagtggtag aagccgtagc ccatcacgtg gctcagcagg taggggctga aggtcagggc
      121 gcctttggtg ctcttcatct tgttggtcat gcggccctgc tcgggggtgc cctctccgcc
      181 gcccaccagc tcgaactcca cgccgttcag ggtgccggtg atgcggcact cgatcttcat
      241 ggcgggcata gaacctccac ctccaccagc gtaatctgga acatcgtatg ggtaacctcc
      301 accgccccct ttatcgtcat cgtctttgta gtccatggtg gcggtaccgt cgactgcaga
      361 attcgaagct tgagctcgag atctgagtcc ggtagcgcta gcggatctga cggttcacta
      421 aaccagctct gcttatatag acctcccacc gtacacgcct accgcccatt tgcgtcaatg
      481 gggcggagtt gttacgacat tttggaaagt cccgttgatt ttggtgccaa aacaaactcc
      541 cattgacgtc aatggggtgg agacttggaa atccccgtga gtcaaaccgc tatccacgcc
      601 cattgatgta ctgccaaaac cgcatcacca tggtaatagc gatgactaat acgtagatgt
      661 actgccaagt aggaaagtcc cataaggtca tgtactgggc ataatgccag gcgggccatt
      721 taccgtcatt gacgtcaata gggggcgtac ttggcatatg atacacttga tgtactgcca
      781 agtgggcagt ttaccgtaaa tactccaccc attgacgtca atggaaagtc cctattggcg
      841 ttactatggg aacatacgtc attattgacg tcaatgggcg ggggtcgttg ggcggtcagc
      901 caggcgggcc atttaccgta agttatgtaa cgcggaactc catatatggg ctatgaacta
      961 atgaccccgt aattgattac tattaataac taatgcatgg cggtaatacg gttatccaca
     1021 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     1081 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     1141 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     1201 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     1261 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     1321 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     1381 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     1441 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     1501 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     1561 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     1621 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     1681 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     1741 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     1801 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta acctgaggct
     1861 atggcagggc ctgccgcccc gacgttggct gcgagccctg ggccttcacc cgaacttggg
     1921 gggtggggtg gggaaaagga agaaacgcgg gcgtattggc cccaatgggg tctcggtggg
     1981 gtatcgacag agtgccagcc ctgggaccga accccgcgtt tatgaacaaa cgacccaaca
     2041 ccgtgcgttt tattctgtct ttttattgcc gtcatagcgc gggttccttc cggtattgtc
     2101 tccttccgtg tttcagttag cctcccccta gggtgggcga agaactccag catgagatcc
     2161 ccgcgctgga ggatcatcca gccggcgtcc cggaaaacga ttccgaagcc caacctttca
     2221 tagaaggcgg cggtggaatc gaaatctcgt gatggcaggt tgggcgtcgc ttggtcggtc
     2281 atttcgaacc ccagagtccc gctcagaaga actcgtcaag aaggcgatag aaggcgatgc
     2341 gctgcgaatc gggagcggcg ataccgtaaa gcacgaggaa gcggtcagcc cattcgccgc
     2401 caagctcttc agcaatatca cgggtagcca acgctatgtc ctgatagcgg tccgccacac
     2461 ccagccggcc acagtcgatg aatccagaaa agcggccatt ttccaccatg atattcggca
     2521 agcaggcatc gccatgggtc acgacgagat cctcgccgtc gggcatgctc gccttgagcc
     2581 tggcgaacag ttcggctggc gcgagcccct gatgctcttc gtccagatca tcctgatcga
     2641 caagaccggc ttccatccga gtacgtgctc gctcgatgcg atgtttcgct tggtggtcga
     2701 atgggcaggt agccggatca agcgtatgca gccgccgcat tgcatcagcc atgatggata
     2761 ctttctcggc aggagcaagg tgagatgaca ggagatcctg ccccggcact tcgcccaata
     2821 gcagccagtc ccttcccgct tcagtgacaa cgtcgagcac agctgcgcaa ggaacgcccg
     2881 tcgtggccag ccacgatagc cgcgctgcct cgtcttgcag ttcattcagg gcaccggaca
     2941 ggtcggtctt gacaaaaaga accgggcgcc cctgcgctga cagccggaac acggcggcat
     3001 cagagcagcc gattgtctgt tgtgcccagt catagccgaa tagcctctcc acccaagcgg
     3061 ccggagaacc tgcgtgcaat ccatcttgtt caatcatgcg aaacgatcct catcctgtct
     3121 cttgatcgat ctttgcaaaa gcctaggcct ccaaaaaagc ctcctcacta cttctggaat
     3181 agctcagagg ccgaggcggc ctcggcctct gcataaataa aaaaaattag tcagccatgg
     3241 ggcggagaat gggcggaact gggcggagtt aggggcggga tgggcggagt taggggcggg
     3301 actatggttg ctgactaatt gagatgcatg ctttgcatac ttctgcctgc tggggagcct
     3361 ggggactttc cacacctggt tgctgactaa ttgagatgca tgctttgcat acttctgcct
     3421 gctggggagc ctggggactt tccacaccct aactgacaca cattccacag ctggttcttt
     3481 ccgcctcagg actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     3541 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     3601 ttccccgaaa agtgccacct gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     3661 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     3721 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     3781 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     3841 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     3901 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     3961 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     4021 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttacg
     4081 ccttaagata cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct
     4141 ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac
     4201 aagttaacaa caacaattgc attcatttta tgtttcaggt tcagggggag gtgtgggagg
     4261 ttttttaaag caagtaaaac ctctacaaat gtggtatggc tgattatgat ctagagtcgc
     4321 ggccgcgatt acaatttgga ctttccgccc ttcttggcct ttatgaggat ctctctgatt
     4381 tttcttgcgt cgagttttcc ggtaagacct ttcggtactt cgtccacaaa cacaactcct
     4441 ccgcgcaact ttttcgcggt tgttacttga ctggcgacgt aatccacgat ctctttttcc
     4501 gtcatcgtct ttccgtgctc caaaacaaca acggcggcgg gaagttcacc ggcgtcatcg
     4561 tcgggaagac ctgccacgcc cgcgtcgaag atgttggggt gttgtaacaa tatcgactcc
     4621 aattcagcgg gggccacctg gtatcctttg tatttaatta aagacttcaa gcggtcaact
     4681 atgaagaagt gttcgtcttc gtcccagtaa gctatgtctc cagaatgtag ccatccatcc
     4741 ttgtcaatca aggcgttggt cgcttccgga ttgtttacat aaccggacat aatcataggt
     4801 cctctgacac ataattcgcc tctctgatta acgcccagcg ttttcccggt atccagatcc
     4861 acaaccttcg cttcaaaaaa tggaacaact ttaccgaccg cgcccggttt atcatccccc
     4921 tcgggtgtaa tcagaatagc tgatgtagtc tcagtgagcc catatccttg tcgtatccct
     4981 ggaagatgga agcgttttgc aaccgcctcc ccgacttctt tcgaaagagg tgcgccccca
     5041 gaagcaattt cgtgtaaatt agataaatcg tatttgtcaa tcagagtgct tttggcgaag
     5101 aatgaaaata gggttggtac tagcaacgca ctttgaattt tgtaatcctg aagggatcgt
     5161 aaaaacagct cttcttcaaa tctatacatt aagacgactc gaaatccaca tatcaaatat
     5221 ccgagtgtag taaacattcc aaaaccgtga tggaatggaa caacacttaa aatcgcagta
     5281 tccggaatga tttgattgcc aaaaatagga tctctggcat gcgagaatct gacgcaggca
     5341 gttctatgcg gaagggccac acccttaggt aacccagtag atccagagga gttcattatc
     5401 agtgcaattg ttttgtcacg atcaaaggac tctggtacaa aatcgtattc attaaaaccg
     5461 ggaggtagat gagatgtgac gaacgtgtac atcgactgaa atccctggta atccgtttta
     5521 gaatccatga taataatttt ctggattatt ggtaattttt tttgcacgtt caaaattttt
     5581 tgcaacccct ttttggaaac aaacactacg gtaggctgcg aaatgttcat actgttgagc
     5641 aattcacgtt cattataaat gtcgttcgcg ggcgcaactg caactccgat aaataacgcg
     5701 cccaacaccg gcataaagaa ttgaagagag ttttcactgc atacgacgat tctgtgattt
     5761 gtattcagcc catatcgttt catagcttct gccaaccgaa cggacatttc gaagtattcc
     5821 gcgtacgtga tgttcacctc gatatgtgca tctgtaaaag caattgttcc aggaaccagg
     5881 gcgtatctct tcatagcctt atgcagttgc tctccagcgg ttccatcctc tagtggatag
     5941 aatggcgccg ggcctttctt tatgtttttg gcgtcttcca taccggaagg gccgggattc
     6001 tcctccacgt caccgcatgt tagaagactt cctctgccct caccggtcac attgatcctt
     6061 taagcagaag cacaggctgc agggtgacgg tccatcccgc tctcctgggc acaagacatg
     6121 ggcagcgtgc catcatcctg cgccgccacc gccggcggga agccatggct gatatctcga
     6181 gatctggcga aggcgatggg ggtcttgaag gcgtgctggt actccacgat gcccagctcg
     6241 gtgttgctgt gcagctcctc cacgcggcgg aaggcgaaca tggggccccc gttctgcagg
     6301 atgctggggt ggatggcgct cttgaagtgc atgtggctgt ccaccacgaa gctgtagtag
     6361 ccgccgtcgc gcaggctgaa ggtgcgggcg aagctgccca ccagcacgtt atcgcccatg
     6421 gggtgcaggt gctccacggt ggcgttgctg cggatgatct tgtcggtgaa gatcacgctg
     6481 tcctcgggga agccggtgcc caccaccttg aagtcgccga tcacgcggcc ggcctcgtag
     6541 cggtagctga agctcacgtg cagcacgccg ccgtcctcgt acttctcgat gcgggtgttg
     6601 gt
//
